The present invention relates generally to the amplification of nucleic acids. More specifically, the present invention facilitates the amplification of mRNA for a variety of end uses.
Those skilled in the art know there are many ways to synthesize first strand cDNA from mRNA (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987)). For example, the first strand cDNA can be synthesized by a reverse transcriptase with a primer. Reverse transcriptases are readily available from many sources and those skilled in the art will know what reverse transcriptase to use for their specific purposes.
Other suitable amplification methods include the ligase chain reaction (LCR) e.g., Wu and Wallace, Genomics 4, 560 (1989) and Landegren et al., Science 241, 1077 (1988), Burg, U.S. Pat. Nos. 5,437,990, 5,215,899, 5,466,586, 4,357,421, Gubler et al., 1985, Biochemica et Biophysica Acta, Displacement Synthesis of Globin Complementary DNA: Evidence for Sequence Amplification, transcription amplification, Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989), self-sustained sequence replication, Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO 88/10315 and WO 90/06995 and nucleic acid based sequence amplification (NABSA). The latter two amplification methods include isothermal reactions based on isothermal transcription, which produce both single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively. Second strand priming can occur by hairpin loop formation, RNAse H digestion products, and the 3xe2x80x2 end of any nucleic acid present in a reaction capable of forming an extensible complex with the first strand DNA.
Common amplification procedures can encounter problems when adding transcriptional promoters. When promoters are incorporated early during amplification, partial degradation is often observed as a result of the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of DNA polymerases. Second, amplification that relies upon promoter sequences being incorporated into the primer allows for only one promoter orientation, sense or antisense. Third, the use of short primers (less than 50 bases) ensures greater product reliability than use of longer primers (greater than 50 bases) such as those that include transcriptional promoters.
The present invention provides a method for the amplification of nucleic acids that may comprise synthesizing double-stranded DNA from a single-stranded DNA population, and producing multiple copies of RNA from the double-stranded DNA More specifically, the method comprises contacting a mRNA having a poly dA tail with a primer comprising poly d(T) and a second sequence; generating a first cDNA strand from the mRNA strand by extending the primer by reverse transcriptase and the appropriate nucleotides under the appropriate conditions, which creates a RNA:DNA duplex; denaturing the RNA:DNA duplex; forming a double stranded DNA; denaturing the double stranded DNA to form a single stranded DNA and adding a promoter to the single stranded DNA, the promoter comprising a complement to the second sequence and a full or partial RNA promoter sufficient to form a functional promoter when the promoter is hybridized to the single stranded DNA; forming a double stranded DNA promoter region by adding the appropriate reagents; and, producing multiple copies of RNA from the DNA strand comprising the promoter. Preferably, the promoter is blocked from 3xe2x80x2 extension.
Among other factors, the present invention provides a new method for amplification of nucleic acids. Additionally, the present method is an amplification method in which a promoter is protected from degradation throughout the method.
The present invention also preferably provides methods, which may further comprise contacting the multiple copies of RNA with a solid support comprising nucleic acid probes, and detecting the presence or absence of hybridization of the RNA to the nucleic acid probes on the solid support. In a preferred embodiment, the solid support, which may comprise nucleic acid probes, can be selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.
In yet another preferred embodiment, the invention relates to a kit comprising reagents and instructions for the amplification of mRNA. Preferably, the kit includes a reaction vessel containing one or more reagents in concentrated form, where the reagent may be an enzyme or enzyme mixture. The kit also includes a container, instructions for use, a promoter which comprises a poly d(T) sequence operably linked to a second sequence, a promoter comprising the second sequence or its equivalent and a sequence that will complete a promoter when annealed to a target and formed into a double stranded DNA. Preferably, the promoter is blocked from extending in the 3xe2x80x2 direction.